www.impactjournals.com/oncotarget/
Oncotarget, 2016, Vol. 7, (No. 49), pp: 80415-80425 Research Paper
Gli promotes epithelial-mesenchymal transition in human lung adenocarcinomas Hui Li1,*, Dongsheng Yue1,2,*, Joy Q. Jin1, Gavitt A. Woodard1, Bhairavi Tolani1, Thomas M. Luh1, Etienne Giroux-Leprieur1, Minli Mo3, Zhao Chen3, Juanjuan Che4, Zhenfa Zhang2, Yong Zhou1, Lei Wang1,5, Xishan Hao2, David Jablons1, Changli Wang2, Biao He1 1
Thoracic Oncology Program, Department of Surgery, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94115, USA
2
Department of Lung Cancer, Lung Cancer Center, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China
3
Beijing ACCB Biotech Ltd., Beijing 100084, China
4
Department of Oncology, Beijing Friendship Hospital of Capital Medical University, Beijing 100050, China
5
Department of Thoracic Surgery, Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, China
*
These authors have contributed equally to this work
Correspondence to: Biao He, email: [email protected] Changli Wang, email: [email protected] Keywords: sonic hedgehog, gli, epithelial-mesenchymal transition, lung cancer, adenocarcinoma Received: May 09, 2016 Accepted: July 28, 2016 Published: August 12, 2016
ABSTRACT Adenocarcinoma is the most common type of lung cancer. Epithelial-mesenchymal transition (EMT) is required for tumor invasion/metastasis and the components that control this process are potential therapeutic targets. This study we examined the role of Gli in lung adenocarcinoma and whether its activation regulates metastasis through EMT in lung adenocarcinoma. We found that tumors with high Gli expression had significantly lower E-Cadherin expression in two independent cohorts of patients with lung adenocarcinoma that we studied. In vitro up-regulation of SHh resulted in increased cell migration while small molecule inhibitors of Smo or Gli significantly reduced cell mobility both in a wound healing assay and in a 3D cell invasion assay. Inhibition of Gli in vivo decreased tumor growth and induced an increase in E-Cadherin expression. Our results indicate that Gli may be critical for lung adenocarcinoma metastasis and that a novel Gli inhibitor shows promise as a therapeutic agent by preventing cell migration and invasion in vitro and significantly reducing tumor growth and increasing E-Cadherin expression in vivo.
needed [3, 4]. Once patients have developed metastatic disease only 15% will be alive after one year and there are virtually no longer term survivors [5], underlying the importance in developing treatment strategies which target the mechanisms leading to tumor invasion and metastasis. Epithelial-mesenchymal transition (EMT) is a process in which cells lose their cell-cell adhesive properties and gain migratory and invasive potential. EMT is essential for events in embryonic development, wound healing, fibrosis, and in cancer progression and metastasis. It has been well established that epithelial cancer cells undergo EMT for tumor progression and
INTRODUCTION Lung cancer remains the leading cause of cancerrelated mortality in the United States and worldwide with an overall 5 year survival rate of 19.3% [1]. Adenocarcinoma is the most common form of lung cancer and accounts for 38% of all newly diagnosed lung cancers [2]. Recent advancements in targeted therapies such as EGFR tyrosine kinase inhibitors and ALK inhibitors have led to modest improvements in survival times for certain subgroups of patients with lung adenocarcinoma, however, additional therapeutic strategies are desperately
www.impactjournals.com/oncotarget
80415
Oncotarget
in order to invade local tissue and metastasize [6, 7]. EMT process is regulated by various signaling pathways including Sonic Hedgehog (SHh) which lead to many complex changes in the expression and function of proteins. The loss of the cell-to-cell adhesive protein E-Cadherin, which is inactivated in many cancers, is a fundamental event in EMT. During EMT, E-Cadherin is replaced with N-Cadherin, which enhances the motility of tumor cells [8]. EMT confers on cells the critical traits required for seeding metastasis and for developing the stem cell properties that allow for launching of new cancer cell colonies [9]. Acquisition of EMT features has been associated with poor prognosis as well as resistance to chemotherapy [10–12]. Further knowledge of the process of EMT can improve our understanding of tumor recurrence and metastasis and identify potential therapeutic targets. The role of SHh pathway in cancer development is suspected to be via the transformation of adult stem cells into cancer stem cells. Activated SHh has been implicated in tumorigenesis and metastasis in multiple types of cancers including lung, brain, breast, prostate, and skin. In the canonical SHh pathway, the absence of the SHh ligand leads the transmembrane receptor Patched (Ptch) to inhibit the transmembrane receptor Smoothened (Smo). Inhibited Smo causes cleavage of Gli to the N-terminal repressor form. Therefore when SHh binds to Ptch, the inhibitory effect on Smo is released and active full length Gli is transported into the nucleus and activates transcription of Gli-dependent target genes such as Gli1, Ptch1, CyclinD1 and Wnt [13–16]. However, noncanonical Gli activation independent of SHh, has been shown in many cancer cells types [17, 18], and there is evidence for mechanisms of Gli activation independent of SHh, stimulated by other oncogenic signaling pathways such as transforming growth factor β (TGFβ), epidermal growth factor receptor (EGFR), RAS and AKT/PI3K pathways [19–23]. As Gli transcription factors constitute the final effectors of the SHh pathway, and are implicated in multiple other oncogenic signaling pathways, they represent an important downstream target for potential cancer therapeutics [17]. The relationship of SHh pathway to EMT has not been previously studied in lung adenocarcinomas and the existing data from other solid tumors is controversial. There is a growing body of literature that shows that SHh/Gli inhibition blocks EMT, however the exact mechanisms remain to be elucidated. Some studies in melanoma and pancreatic cancers have suggested that Gli facilitates cancer cell migration and invasion via E-Cadherin [24, 25]. In lung squamous cell cancer (SCC) and in hepatocellular carcinoma, Gli expression has been shown to be inversely correlated with E-Cadherin expression and in lung SCC inhibition of the SHh pathway increases E-Cadherin expression [26, 27]. In hepatocellular cancer, Gli1 over-expression www.impactjournals.com/oncotarget
is correlated with capsular invasion, advanced tumor stage, vascular invasion and intrahepatic metastasis and interfering with Gli transcription suppresses cell migration by down-regulating matrix metalloprotease (MMP)-2 and MMP-9 [28]. In vitro down-regulation of Gli1 with siRNA reduced hepatoceullular cancer cell invasion and increased E-Cadherin expression [27]. However there is some conflicting data that showed inhibition of Gli promoted EMT in pancreatic cancer [29]. We have recently demonstrated increased SHh signaling in lung SCC and that Gli1 expression is inversely correlated with the EMT marker E-Cadherin. Inhibition of the SHh pathway up-regulates E-Cadherin expression and suppresses lung SCC cell migration [26]. Here, we report the Gli activation in two cohorts of patients with lung adenocarcinomas and show that Gli1 and EMT markers are inversely correlated in lung adenocarcinoma. Inhibition of Gli suppresses migration of lung adenocarcinoma cells and up-regulates E-Cadherin expression in vitro. We also demonstrate a reduction in lung adenocarcinoma growth in vivo by a small molecule Gli inhibitor.
RESULTS Gli expression inversely correlates with E-Cadherin expression in lung adenocarcinoma We investigated the expression of Gli proteins and E-Cadherin in lung adenocarcinoma patient tissues from the Lung Cancer Center at Tianjin Medical University Cancer Institute and Hospital, Tianjin and the Thoracic Oncology Program at University of California, San Francisco. The expression of Gli1, Gli2 and E-Cadherin was evaluated by immunohistochemistry (IHC) with 68 formalin-fixed, paraffin-embedded tissue specimens from the Tianjin cohort. Clinical and demographic information from the Tianjin cohort is summarized in Table 1. Tumor samples with high Gli1 or Gli2 expression showed lower E-Cadherin expression while low Gli expression showed high expression with an epithelial growth pattern (Figure 1A). The protein expressions of Gli1, Gli2, and E-Cadherin were scored a “high” or “low” expression based on IHC as previously described [26]. Statistical analysis with Kendall’s tau-b correlation tests revealed that both Gli1 and Gli2 were significantly inversely correlated with E-Cadherin expression (p-value 0.01 and 0.03 respectively) (Figure 1B). In addition, we found E-Cadherin exhibited a heterogeneous pattern in some tissue samples, and Gli expression seemed inversely correlated with E-Cadherin within the same specimen. To confirm the inverse correlation between Gli and E-Cadherin, we conducted co-immunofluorescence of Gli2 and E-Cad on 17 tissue samples from UCSF, and 13 of these 17 (76%) samples exhibited a clear inverse 80416
Oncotarget
Table 1: Patient Information Characteristics
Number
Percent
Oncotarget, 2016, Vol. 7, (No. 49), pp: 80415-80425 Research Paper
Gli promotes epithelial-mesenchymal transition in human lung adenocarcinomas Hui Li1,*, Dongsheng Yue1,2,*, Joy Q. Jin1, Gavitt A. Woodard1, Bhairavi Tolani1, Thomas M. Luh1, Etienne Giroux-Leprieur1, Minli Mo3, Zhao Chen3, Juanjuan Che4, Zhenfa Zhang2, Yong Zhou1, Lei Wang1,5, Xishan Hao2, David Jablons1, Changli Wang2, Biao He1 1
Thoracic Oncology Program, Department of Surgery, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94115, USA
2
Department of Lung Cancer, Lung Cancer Center, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China
3
Beijing ACCB Biotech Ltd., Beijing 100084, China
4
Department of Oncology, Beijing Friendship Hospital of Capital Medical University, Beijing 100050, China
5
Department of Thoracic Surgery, Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, China
*
These authors have contributed equally to this work
Correspondence to: Biao He, email: [email protected] Changli Wang, email: [email protected] Keywords: sonic hedgehog, gli, epithelial-mesenchymal transition, lung cancer, adenocarcinoma Received: May 09, 2016 Accepted: July 28, 2016 Published: August 12, 2016
ABSTRACT Adenocarcinoma is the most common type of lung cancer. Epithelial-mesenchymal transition (EMT) is required for tumor invasion/metastasis and the components that control this process are potential therapeutic targets. This study we examined the role of Gli in lung adenocarcinoma and whether its activation regulates metastasis through EMT in lung adenocarcinoma. We found that tumors with high Gli expression had significantly lower E-Cadherin expression in two independent cohorts of patients with lung adenocarcinoma that we studied. In vitro up-regulation of SHh resulted in increased cell migration while small molecule inhibitors of Smo or Gli significantly reduced cell mobility both in a wound healing assay and in a 3D cell invasion assay. Inhibition of Gli in vivo decreased tumor growth and induced an increase in E-Cadherin expression. Our results indicate that Gli may be critical for lung adenocarcinoma metastasis and that a novel Gli inhibitor shows promise as a therapeutic agent by preventing cell migration and invasion in vitro and significantly reducing tumor growth and increasing E-Cadherin expression in vivo.
needed [3, 4]. Once patients have developed metastatic disease only 15% will be alive after one year and there are virtually no longer term survivors [5], underlying the importance in developing treatment strategies which target the mechanisms leading to tumor invasion and metastasis. Epithelial-mesenchymal transition (EMT) is a process in which cells lose their cell-cell adhesive properties and gain migratory and invasive potential. EMT is essential for events in embryonic development, wound healing, fibrosis, and in cancer progression and metastasis. It has been well established that epithelial cancer cells undergo EMT for tumor progression and
INTRODUCTION Lung cancer remains the leading cause of cancerrelated mortality in the United States and worldwide with an overall 5 year survival rate of 19.3% [1]. Adenocarcinoma is the most common form of lung cancer and accounts for 38% of all newly diagnosed lung cancers [2]. Recent advancements in targeted therapies such as EGFR tyrosine kinase inhibitors and ALK inhibitors have led to modest improvements in survival times for certain subgroups of patients with lung adenocarcinoma, however, additional therapeutic strategies are desperately
www.impactjournals.com/oncotarget
80415
Oncotarget
in order to invade local tissue and metastasize [6, 7]. EMT process is regulated by various signaling pathways including Sonic Hedgehog (SHh) which lead to many complex changes in the expression and function of proteins. The loss of the cell-to-cell adhesive protein E-Cadherin, which is inactivated in many cancers, is a fundamental event in EMT. During EMT, E-Cadherin is replaced with N-Cadherin, which enhances the motility of tumor cells [8]. EMT confers on cells the critical traits required for seeding metastasis and for developing the stem cell properties that allow for launching of new cancer cell colonies [9]. Acquisition of EMT features has been associated with poor prognosis as well as resistance to chemotherapy [10–12]. Further knowledge of the process of EMT can improve our understanding of tumor recurrence and metastasis and identify potential therapeutic targets. The role of SHh pathway in cancer development is suspected to be via the transformation of adult stem cells into cancer stem cells. Activated SHh has been implicated in tumorigenesis and metastasis in multiple types of cancers including lung, brain, breast, prostate, and skin. In the canonical SHh pathway, the absence of the SHh ligand leads the transmembrane receptor Patched (Ptch) to inhibit the transmembrane receptor Smoothened (Smo). Inhibited Smo causes cleavage of Gli to the N-terminal repressor form. Therefore when SHh binds to Ptch, the inhibitory effect on Smo is released and active full length Gli is transported into the nucleus and activates transcription of Gli-dependent target genes such as Gli1, Ptch1, CyclinD1 and Wnt [13–16]. However, noncanonical Gli activation independent of SHh, has been shown in many cancer cells types [17, 18], and there is evidence for mechanisms of Gli activation independent of SHh, stimulated by other oncogenic signaling pathways such as transforming growth factor β (TGFβ), epidermal growth factor receptor (EGFR), RAS and AKT/PI3K pathways [19–23]. As Gli transcription factors constitute the final effectors of the SHh pathway, and are implicated in multiple other oncogenic signaling pathways, they represent an important downstream target for potential cancer therapeutics [17]. The relationship of SHh pathway to EMT has not been previously studied in lung adenocarcinomas and the existing data from other solid tumors is controversial. There is a growing body of literature that shows that SHh/Gli inhibition blocks EMT, however the exact mechanisms remain to be elucidated. Some studies in melanoma and pancreatic cancers have suggested that Gli facilitates cancer cell migration and invasion via E-Cadherin [24, 25]. In lung squamous cell cancer (SCC) and in hepatocellular carcinoma, Gli expression has been shown to be inversely correlated with E-Cadherin expression and in lung SCC inhibition of the SHh pathway increases E-Cadherin expression [26, 27]. In hepatocellular cancer, Gli1 over-expression www.impactjournals.com/oncotarget
is correlated with capsular invasion, advanced tumor stage, vascular invasion and intrahepatic metastasis and interfering with Gli transcription suppresses cell migration by down-regulating matrix metalloprotease (MMP)-2 and MMP-9 [28]. In vitro down-regulation of Gli1 with siRNA reduced hepatoceullular cancer cell invasion and increased E-Cadherin expression [27]. However there is some conflicting data that showed inhibition of Gli promoted EMT in pancreatic cancer [29]. We have recently demonstrated increased SHh signaling in lung SCC and that Gli1 expression is inversely correlated with the EMT marker E-Cadherin. Inhibition of the SHh pathway up-regulates E-Cadherin expression and suppresses lung SCC cell migration [26]. Here, we report the Gli activation in two cohorts of patients with lung adenocarcinomas and show that Gli1 and EMT markers are inversely correlated in lung adenocarcinoma. Inhibition of Gli suppresses migration of lung adenocarcinoma cells and up-regulates E-Cadherin expression in vitro. We also demonstrate a reduction in lung adenocarcinoma growth in vivo by a small molecule Gli inhibitor.
RESULTS Gli expression inversely correlates with E-Cadherin expression in lung adenocarcinoma We investigated the expression of Gli proteins and E-Cadherin in lung adenocarcinoma patient tissues from the Lung Cancer Center at Tianjin Medical University Cancer Institute and Hospital, Tianjin and the Thoracic Oncology Program at University of California, San Francisco. The expression of Gli1, Gli2 and E-Cadherin was evaluated by immunohistochemistry (IHC) with 68 formalin-fixed, paraffin-embedded tissue specimens from the Tianjin cohort. Clinical and demographic information from the Tianjin cohort is summarized in Table 1. Tumor samples with high Gli1 or Gli2 expression showed lower E-Cadherin expression while low Gli expression showed high expression with an epithelial growth pattern (Figure 1A). The protein expressions of Gli1, Gli2, and E-Cadherin were scored a “high” or “low” expression based on IHC as previously described [26]. Statistical analysis with Kendall’s tau-b correlation tests revealed that both Gli1 and Gli2 were significantly inversely correlated with E-Cadherin expression (p-value 0.01 and 0.03 respectively) (Figure 1B). In addition, we found E-Cadherin exhibited a heterogeneous pattern in some tissue samples, and Gli expression seemed inversely correlated with E-Cadherin within the same specimen. To confirm the inverse correlation between Gli and E-Cadherin, we conducted co-immunofluorescence of Gli2 and E-Cad on 17 tissue samples from UCSF, and 13 of these 17 (76%) samples exhibited a clear inverse 80416
Oncotarget
Table 1: Patient Information Characteristics
Number
Percent
